Physostigmine is a tertiary amine which can be isolated from the seed of the physostigma venenosum balfour, a plant native to coastal areas of Africa. Physostigmine is soluble in biological fluids and salts of physostigmine, e.g., sulfate and salicylate, have many therapeutic uses. These salts are usually colorless, odorless crystals which gradually turn red upon exposure to air and light. The rate of color change is enhanced in the presence of moisture. Physostigmine salts are soluble in water, but are unstable in alkaline solutions.
Physostigmine is a reversible cholinesterase inhibitor which prevents the hydrolysis of acetylcholine by competing with acetylcholine for attachment to acetylcholinesterase. Acetylcholine is a neurotransmitter stored in vesicles where it is primarily released by nerve impulses. The vesicles migrate towards the terminal synaptic membrane during nerve stimulation and disgorge acetylcholine by exocytosis. Upon release from the cholinergic nerve endings, acetylcholine is inactivated by enzymatic degradation. The inactivation is accomplished by the hydrolysis of acetylcholine by cholinesterase. The specific cholinesterase for acetylcholine, acetylcholinesterase, is quite efficient--one molecule of the enzyme is able to hydrolyze 3.times.10.sup.5 molecules of acetylcholine per minute.
Because a physostigmine-acetylcholinesterase enzyme complex hydrolyzes at a much slower rate than the corresponding acetylcholine-acetylcholinesterase enzyme complex, acetylcholine accumulates at the cholinergic synapses. Due to the reversible nature, i.e., uncoupling, of the physostigmine-acetylcholinesterase complex, physostigmine appears to facilitate the transmission of impulses across the myoneural junction.
There are several clinical uses for reversible cholinesterase inhibiting agents such as physostigmine. Physostigmine is used to improve muscle strength in the symptomatic treatment of mysthenia gravis. Parental physostigmine is also useful in reversing of the effects of nondepolarizing neuromuscular blocking agents, e.g., tubocurarine, metocurine, gallamine or pancuronium, after surgery. Recently, cholinesterase inhibitors have been used in an attempt to reverse certain degenerative disorders of the central nervous system. Moreover, physostigmine may be of value in the treatment of cognitive disorders which involve disturbances of memory. For example, it has been suggested that since cognitive changes observed during the aging process, e.g., Alzheimer's syndrome, may be related to gradual reductions in acetylcholine in various parts of the brain, the administration of physostigmine might help reduce or reverse the observed cognitive changes.
Physostigmine may also be used to block intoxication by irreversible cholinesterase inhibitors. Reversible cholinesterase inhibitors, such as physostigmine, have been proposed as antidotes to nerve agents used in chemical warfare. Many nerve agents are organo-phosphorous compounds which are readily vaporized under normal atmospheric conditions. The extreme toxicity of such organo-phosphorous compounds are related to their short-lived, but irreversible, destruction of the functioning of nerves and organs. By phosphorylating acetylcholinesterase, these organo-phosphorous compounds form stable, irreversible complexes with acetylcholinesterase. The formation of such stable complexes permanently prevents the normal function of acetylcholinesterase, i.e., the termination of acetylcholine actions at synaptic, particularly neuromuscular, junctions. Since the enzyme is completely and permanently prevented from binding with acetylcholine, the acetylcholine quickly accumulates at receptor sites to a degree sufficient to produce loss of function in target nerves and organs.
From animal studies, it has been proposed that nerve agent toxicity can be prevented by the preadministration of a short acting, reversible cholinesterase inhibitor such as physostigmine. The physostigmine would temporarily bind acetylcholinesterase in the tissue which would prevent its phosphorylation by the nerve agents and the resulting irreversible inactivation of the active site of the acetylcholinesterase.
To date, however, results obtained from the administration of cholinesterase inhibitors such as physostigmine to maintain, restore or increase acetylcholine levels in patients, including those with Alzheimer's syndrome, have been equivocal. A primary problem encountered with the clinical use of physostigmine has been that it is toxic at levels very close to those which produce therapeutic results. For example, in certain patients physostigmine has been associated with adverse effects typical of exaggerated responses to parasympathetic stimulation including adverse muscarinic effects such as nausea, vomiting, diarrhea, miosis, excessive salivation and sweating, abdominal cramps, bradycardia, bronchial secretion and bronchospasm. Other side effects of physostigmine include generalized weakness, muscle cramps, fasciculation, hypotension, and, if administered intraveneously, thrombophlebitis. A substantial over administration of physostigmine causes cholinergic crisis leading to death.
Although physostigmine is widely used in clinical medicine, there is no simple and reliable method to measure minute concentrations of the drug in biological fluids. Moreover, relatively little is known about the pharamacokinetic parameters, i.e., the change in concentration at various sites, including absorption, distribution, metabolism and excretion, of physostigmine in man. The dearth of information is due primarily to the lack of a satisfactory analytical method for repeatedly measuring low concentrations of physostigmine in biological fluids.
Presently, a quantitative assay using human blood is available to measure the amount of physostigmine. This assay is based on the duration of cholinesterase inhibition and can detect microgram quantities of physostigmine. A high pressure liquid chromatography (HPLC) method is available which can detect 50 ng of physostigmine in a biological sample. These methods, however, are not sufficiently sensitive for the study of the drug's kinetics using plasma and tissue samples. Tritiated physostigmine has been used to study the pharmacokinetics of the drug in laboratory animals. This method is sensitive but would not be suitable to monitor plasma concentrations of the drug in clinical situations. In fact, all of the presently known methods are insufficient or too expensive and suggest the need for a sensitive, specific, cost effective method for the monitoring of physostigmine concentrations in biological samples.
There are several reasons why such analytical methods for measuring physostigmine in biological fluids have been unsatisfactory. The amounts of physostigmine to be measured are extremely low, which means that any analytical method must be sufficiently sensitive to detect extremely low levels of physostigmine. Moreover, physostigmine undergoes extensive in vitro hydrolysis in biological solutions, particularly in plasma and blood and the method must be specific so that metabolites are not measured as physostigmine.
While radioimmunoassay (RIA) procedures are widely used in clinical and research laboratories to determine the minute quantities of numerous substances in biological fluids, heretobefore an immunoassay for determining the presence of physostigmine in such fluids has been unavailable, primarily because the physostigmine molecule is very small and can not stimulate the immune systems of animals to produce antibodies. The inability to stimulate the immune system is further complicated by the fact that the physostigmine molecule contains few of the functional groups which are usually necessary for a molecule to be linked to a protein immunogen.
Moreover, because of its small size, it was believed that an immunogen for physostigmine might lack sufficient selectivity to avoid cross reactivity with the metabolites of physostigmine and other reversible cholinesterese inhibitors or the metabolites thereof. Since the degradation products are 1000 times less potent than the physostigmine in the inhibition of cholinesterase, it is important to specifically measure the parent compound. The binding of small amounts of metabolites to the antibody could result in significant errors in the measurement of the amount of physostigmine induced anticholinesterase activity present.
It is therefore an object of the present invention to provide for a method for the determination of the amount of physostigmine in biological fluid.
It is another object of the present invention to provide an immunoassay for the determination of the amount of physostigmine in biological fluids.
Yet another object of the present invention is to provide immunogens which after injection into animals will result in the production of antibodies for use in an immunoassay for the determination of physostigmine in biological fluids.
Still a further object of the present invention is to provide monoclonal and polyclonal/antibodies which can be used to determine physostigmine in biological fluids.
A further object of the present invention is to provide for a method of measuring physostigmine in biological fluids which facilitates the study of the pharmacokinetics of physostigmine.
Still another object of the present invention is to provide for a reduction in the toxicity and other adverse side effects which results from the clinical use of physostigmine.
These and other objects of the present invention, as will become more readily apparent hereinafter, are achieved by the invention described herein below.